Lyophilized viral formulations

ABSTRACT

Provided herein are lyophilized viral formulations useful for the stabilization and storage of viruses and methods of preparing these formulations. The lyophilized viral formulations described herein include a virus (e.g., a purified virus) and a non-viral composition including excipients. The formulations can be used, for example, to retain the infectivity or immunogenicity of viruses during periods of storage.

CROSS-REFERENCE TO PRIORITY APPLICATION

This application is a continuation of U.S. application Ser. No.13/309,901, filed Dec. 2, 2011, which is currently pending and whichclaims priority to U.S. Provisional Application No. 61/419,020, filedDec. 2, 2010. These applications are incorporated herein by reference intheir entireties.

BACKGROUND

Viruses are important in several therapeutic applications, including,for example, viral therapy and vaccine generation. In these therapeuticapplications, it can be desirable for the viruses to retain theirinfectivity or immunogenicity. However, viruses often lose infectivityor immunogenicity after extended periods due to less than optimalformulations or unsuitable storage conditions.

SUMMARY

Provided herein are lyophilized viral formulations useful for thestabilization and storage of viruses and methods of preparing theseformulations. The formulations can be used, for example, to preserveviruses (i.e., to retain the infectivity or immunogenicity of viruses)during periods of storage. The formulations can have lower levels ofparticulates, making them more suitable for parenteral infusion orinjection.

The viral formulations described herein include a virus (e.g., apurified virus) and a non-viral composition including excipients. Insome examples, the viral formulations include a purified virus and anon-viral composition comprising mannitol, sorbitol, histidine, andMg²⁺. In these examples, the viral formulations can be lyophilized.Prior to lyophilization, the non-viral composition is a liquid non-viralcomposition further comprising a liquid carrier. The concentration ofsorbitol in the liquid non-viral composition can be less than 3% basedon the weight of the liquid non-viral composition. The liquid non-viralcomposition, excluding the liquid carrier, can be substantially free ofmonovalent cationic salts.

In some examples, the viral formulations comprise a purified virus and anon-viral composition comprising mannitol, sorbitol, histidine, andMg²⁺. In these examples, the viral formulations can be lyophilized.Prior to lyophilization, the non-viral composition can be a liquidnon-viral composition further comprising a liquid carrier. In someexamples, the concentration of sugars in the liquid non-viralcomposition can be less than 7.5% by weight based on the weight of thenon-viral composition.

The combined concentration of mannitol and sorbitol in the liquidnon-viral composition can be less than 10% by weight (e.g., 7% byweight) based on the weight of the liquid non-viral composition. In someexamples, the viral formulation is substantially free of Zn²⁺ and/ortrehalose. Optionally, the non-viral composition further includes anon-ionic surfactant.

The viral formulations described herein can comprise a purified virusand a non-viral composition comprising sucrose, Mg²⁺, and a non-ionicsurfactant. The viral formulations can be lyophilized. In theseexamples, the non-viral composition can be, prior to lyophilization, aliquid non-viral composition further comprising a liquid carrier. Inthese examples, the concentration of sucrose in the liquid non-viralcomposition, prior to lyophilization, is less than 5% based on theweight of the liquid non-viral composition. In these examples, theliquid non-viral compositions, excluding the liquid carrier, can besubstantially free of monovalent cationic salts, non-sucrose polyols,and carboxylates.

Further, the viral formulations provided herein can consist essentiallyof a purified virus and a non-viral composition comprising sucrose,Mg²⁺, and a non-ionic surfactant. In these examples, the viralformulations can be lyophilized. Prior to lyophilization, the non-viralcomposition can be a liquid non-viral composition further comprising aliquid carrier. The concentration of sucrose in the liquid non-viralcomposition can be less than 5% based on the weight of the liquidnon-viral composition.

Optionally, Mg²⁺ is present as magnesium chloride. The non-ionicsurfactant is optionally polysorbate 80. The liquid carrier can be anaqueous carrier such as water. The virus included in the lyophilizedviral formulations described herein can be, for example, an oncolyticvirus and/or a non-enveloped virus. Provided herein is a lyophilizedformulation in which the virus is a reovirus such as a mammalianreovirus. An example of a mammalian reovirus is a human reovirus, suchas a serotype 3 virus (e.g., the Dearing strain reovirus). The reovirusis optionally a recombinant reovirus, a reassorted reovirus, or IDAC#190907-01.

Optionally, the viral formulations are stable at a temperature at aboutambient temperature for a period of time (e.g., at least one day).Optionally, the viral formulations are stable at a temperature of about4° C. or lower for at least three months (e.g., at least six months, atleast twelve months, at least eighteen months, or any amount of timegreater than three months). Optionally, the viral formulations aresuitable for reconstitution before administration. Reconstituted viralformulations can be further diluted to achieve a preferred dose foradministration.

Also provided herein are methods of making the viral formulations. Themethods include providing the virus, combining the virus and a liquidnon-viral composition (including the excipients as described herein anda liquid carrier) to form a liquid viral formulation, and lyophilizingthe liquid viral formulation. Lyophilizing the liquid viral formulationoptionally comprises the steps of freezing the liquid viral formulationto a temperature lower than 0° C. to form a frozen viral formulation andapplying a vacuum to the frozen viral formulation. In some examples, themethods further comprise reconstituting the lyophilized viralformulation (e.g., dissolving or suspending the lyophilized viralformulation in a medium). The methods optionally further comprise addinga non-ionic surfactant to the composition. Viral formulations preparedaccording to these methods are also described herein.

Further described herein are methods of preserving or stabilizing avirus. The methods include preparing a lyophilized viral formulation asdescribed herein and storing the lyophilized viral formulation. In someexamples, the virus is stored at a temperature at or below ambienttemperature. For example, the temperature can be ambient temperature orfrom 2° C. to 8° C. (e.g., 4° C.). In some examples, the temperature is−20° C. or from −60° C. to −80° C.

Additionally described herein are methods of preparing viralformulations with low levels of particulates prior to lyophilization,e.g., non-aggregating viral formulations. Optionally, the viralformulations comprise fewer than 6,000 particles having a particle sizeof 10 microns or greater per container (e.g., fewer than 3,000particles, fewer than 2,000 particles, fewer than 1,000 particles, fewerthan 500 particles, fewer than 300 particles, or fewer than 100particles of 10 microns or greater per container). Optionally, the viralformulations comprise fewer than 600 particles having a particle size of25 microns or greater (e.g., fewer than 500 particles, fewer than 400particles, fewer than 300 particles, fewer than 200 particles, fewerthan 100 particles, fewer than 50 particles, or fewer than 10 particlesof 25 microns or greater per container). The methods include preparingthe viral formulation as described herein and then lyophilizing thecomposition to prepare a lyophilized viral formulation. Optionally, thelyophilized viral formulation can be reconstituted and the reconstitutedviral formulations can be suitable for administration by parenteralinfusion or injection.

The details of one or more aspects are set forth in the accompanyingdrawings and the description below. Other features, objects, andadvantages will be apparent from the description and drawings, and fromthe claims.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a bar graph showing the median tissue culture infective dose,expressed as TCID₅₀ per milliliter, for lyophilized reovirusformulations prior to storage (t=0) at ambient temperature and after 3(t=3 m), 6.5 (t=6.5 m), 12 (t=12 m), and 18.5 (t=18.5 m) months ofstorage at ambient temperature, 2-8° C., −20° C., and −80° C. For eachformulation, from left to right: t=0 at ambient temperature; t=18.5 m at−80° C.; t=3 m at −20° C.; t=6.5 m at −20° C.; t=12 m at −20° C.; t=18.5m at −20° C.; t=3 m at 2-8° C.; t=6.5 m at 2-8° C.; t=12 m at 2-8° C.;t=18.5 m at 2-8° C.; t=3 m at ambient temperature; t=6.5 m at ambienttemperature; t=12 m at ambient temperature. For Formulation 1, phosphatebuffered saline-eluted reovirus was diluted in 30 mg/mL mannitol, 20mg/mL histidine, 0.01% v/v polysorbate 80, 20 mg/mL sorbitol, and 2 mMMgCl₂, to a viral titer of 3×10¹⁰ TCID₅₀/mL. For Formulation 2,phosphate buffered saline-eluted reovirus was diluted in 40 mg/mLsucrose, 0.05% v/v polysorbate 80, and 2 mM MgCl₂, to a viral titer of3×10¹⁰ TCID₅₀/mL.

FIG. 2 is a bar graph showing the recovery of infectious particles basedon the percentage of median tissue culture infective dose, expressed asTCID₅₀ per milliliter, for lyophilized reovirus formulations prior tostorage (t=0) at ambient temperature and after 3 (t=3 m), 6.5 (t=6.5 m),12 (t=12 m), and 18.5 (t=18.5 m) months of storage at ambienttemperature, 2-8° C., −20° C., and −80° C. For each formulation, fromleft to right: t=0 at ambient temperature; t=18.5 m at −80° C.; t=3 m at−20° C.; t=6.5 m at −20° C.; t=12 m at −20° C.; t=18.5 m at −20° C.; t=3m at 2-8° C.; t=6.5 m at 2-8° C.; t=12 m at 2-8° C.; t=18.5 m at 2-8°C.; t=3 m at ambient temperature; t=6.5 m at ambient temperature; t=12 mat ambient temperature.

FIG. 3 is a bar graph showing the evolution of the normalized totalviral titers measured by HPLC, expressed as viral particles permilliliter, for lyophilized reovirus Formulation 1 prior to storage(t=0) at ambient temperature and after 2 weeks (time=2 wks), 1 month(time=1 m), 2 months (time=2 m), 3 months (time=3 m), 6.5 months(time=6.5 m), 12 months (time=12 m), and 18.5 months (time=18.5 m) ofstorage at 37° C., ambient temperature, 2-8° C., −20° C., and −80° C.For each temperature, from left to right: time=0, time=2 wks, time=1 m,time=2 m, time=3 m, time=6.5 m, time=12 m, and time=18.5 m.

FIG. 4 is a bar graph showing the evolution of the normalized totalviral titers measured by HPLC, expressed as viral particles permilliliter, for lyophilized reovirus Formulation 2 prior to storage(t=0) at ambient temperature and after 2 weeks (time=2 wks), 1 month(time=1 m), 2 months (time=2 m), 3 months (time=3 m), 6.5 months(time=6.5 m), 12 months (time=12 m), and 18.5 months (time=18.5 m) ofstorage at 37° C., ambient temperature, 2-8° C., −20° C., and −80° C.For each temperature, from left to right: time=0, time=2 wks, time=1 m,time=2 m, time=3 m, time=6.5 m, time=12 m, and time=18.5 m. The samplestored for 3 months at −20° C., indicated with an asterisk (*), is froma repeat analysis performed three days after the other storagetemperature samples.

DETAILED DESCRIPTION

Described herein are viral formulations useful for the storage ofviruses and methods of preparing these formulations. The formulationscan be used, for example, to retain the infectivity or immunogenicity ofviruses during periods of storage. The lyophilized viral formulationsdescribed herein include a virus and a non-viral composition includingexcipients.

Viruses for use in the lyophilized formulations described herein includeenveloped and non-enveloped viruses. The enveloped and non-envelopedviruses can be DNA viruses, RNA viruses, or retroviruses. Optionally,the virus for use in the formulations described herein is anon-enveloped virus. Non-enveloped viruses include, for example, virusesbelonging to the families of Adenoviridae (e.g., adenovirus),Picornaviridae (e.g., polio virus), Reoviridae (e.g., reovirus),Papillomaviridae (e.g., papilloma virus), Polyomaviridae (e.g.,polyomavirus), Parvoviridae (e.g., Kilham rat virus), and Iridoviridae(e.g., tipula iridescent virus).

Optionally, the virus is an oncolytic virus. Suitable viruses for use inthe formulations and methods described herein include, but are notlimited to, myoviridae, siphoviridae, podoviridae, tectiviridae,corticoviridae, plasmaviridae, lipothrixviridae, fuselloviridae,poxviridae, iridoviridae, phycodnaviridae, baculoviridae, herpesviridae,adenoviridae, papovaviridae, polydnaviridae, inoviridae, microviridae,geminiviridae, circoviridae, parvoviridae, hepadnaviridae, retroviridae,cystoviridae, reoviridae, birnaviridae, paramyxoviridae, rhabdoviridae,Filoviridae, orthomyxoviridae, bunyaviridae, arenaviridae, leviviridae,picornaviridae, sequiviridae, comoviridae, potyviridae, caliciviridae,astroviridae, nodaviridae, tetraviridae, tombusviridae, coronaviridae,flaviviridae, togaviridae, barnaviridae, and bornaviridae viruses.

The lyophilized formulations optionally include a reovirus. As usedherein, reovirus refers to any virus classified in the reovirus genus,including naturally occurring and recombinant reoviruses. Reoviruses areviruses with a double-stranded, segmented RNA genome. The virionsmeasure 60-80 nm in diameter and possess two icosahedral, concentriccapsid shells. The genome consists of double-stranded RNA in 10-12discrete segments with a total genome size of 16-27 kilobase pairs(kbp). The individual RNA segments vary in size. Three distinct butrelated types of reovirus have been recovered from many species. Allthree types share a common complement-fixing antigen. The human reovirusconsists of three serotypes: type 1 (strain Lang or T1L), type 2 (strainJones, T2J), and type 3 (strain Dearing or strain Abney, T3D or T3A).

As described above, the reovirus can be a recombinant reovirus, whichcan be naturally occurring or non-naturally occurring. The reovirus isdescribed as naturally occurring when it can be isolated from a sourcein nature and has not been intentionally modified by humans in thelaboratory. For example, the reovirus can be from a field source (i.e.,from a human who has been infected with the reovirus). The reovirus mayalso be selected or mutagenized for enhanced activity (e.g., oncolyticactivity). Examples of specific reovirus can be found, for example, inU.S. Patent Application Publication Nos. 2008/0226602 and 2008/0292594.

The reovirus may be modified but still capable of lytically infecting amammalian cell having an active ras pathway. The reovirus may bechemically or biochemically pretreated (e.g., by treatment with aprotease, such as chymotrypsin or trypsin) prior to administration tothe proliferating cells. Pretreatment with a protease removes the outercoat or capsid of the virus and may increase the infectivity of thevirus. The reovirus may be coated in a liposome or micelle (Chandran andNibert, Journal of Virology, 72(1):467-75 (1998)). For example, thevirion may be treated with chymotrypsin in the presence ofmicelle-forming concentrations of alkyl sulfate surfactants to generatea new infectious subviral particle (ISVP).

The reovirus can be a recombinant or reassortant reovirus resulting fromthe recombination/reassortment of genomic segments from two or moregenetically distinct reoviruses. Recombination/reassortment of reovirusgenomic segments may occur in nature following infection of a hostorganism with at least two genetically distinct reoviruses. Recombinantvirions can also be generated in cell culture, for example, byco-infection of permissive host cells with genetically distinctreoviruses. Accordingly, the recombinant reovirus for use in theformulations described herein can result from reassortment of genomesegments from two or more genetically distinct reoviruses, including butnot limited to, human reovirus, such as type 1 (e.g., strain Lang), type2 (e.g., strain Jones), and type 3 (e.g., strain Dearing or strainAbney), non-human mammalian reoviruses, or avian reovirus. In someexamples, the recombinant reoviruses can result from reassortment ofgenome segments from two or more genetically distinct reoviruses whereinat least one parental virus is genetically engineered, comprises one ormore chemically synthesized genomic segments, has been treated withchemical or physical mutagens, or is itself the result of arecombination event. The recombinant reovirus can undergo recombination,for example, in the presence of chemical mutagens, including but notlimited to dimethyl sulfate and ethidium bromide, or physical mutagens,including but not limited to ultraviolet light and other forms ofradiation.

Other examples of suitable recombinant reoviruses include those thatcomprise deletions or duplications in one or more genome segments, thatcomprise additional genetic information as a result of recombinationwith a host cell genome, or that comprise synthetic genes. The reoviruscan also be modified by incorporation of mutated coat proteins, such asfor example σ1, into the virion outer capsid. The proteins can bemutated by replacement, insertion, or deletion. Replacement includes theinsertion of different amino acids in place of the native amino acids.Insertions include the insertion of additional amino acid residues intothe protein at one or more locations. Deletions include deletions of oneor more amino acid residues in the protein. Such mutations can begenerated by methods known in the art. For example, oligonucleotide sitedirected mutagenesis of the gene encoding for one of the coat proteinscan result in the generation of the desired mutant coat protein. In oneembodiment, the reovirus is IDAC #190907-01.

The viruses for use in the lyophilized formulations described herein canbe purified viruses. As used herein, purified viruses refer to virusesthat have been separated from cellular components that naturallyaccompany them. Typically, viruses are considered purified when they areat least 70% (e.g., at least 75%, at least 80%, at least 85%, at least90%, at least 95%, or at least 99%) by dry weight, free from theproteins and other cellular components with which they are naturallyassociated. The viruses can be purified, for example, according to themethods described in U.S. Patent Application Publication Nos.2002/0168764; 2004/0005693; 2005/0095692; 2006/0088869; and2007/0269856, which are incorporated herein by reference in theirentireties. For example, the virus can be separated from other particlesusing the techniques of density gradient centrifugation,ultrafiltration, diafiltration, ion exchange chromatography, sizeexclusion chromatography, high performance liquid chromatography, orcombinations of these.

As discussed above, the viral formulations include a non-viralcomposition comprising excipients. Optionally, at least two excipients(e.g., two, three, or more excipients) are included in the non-viralcomposition. Excipients for use in the non-viral compositions include,but are not limited to, sugars, amino acids, divalent cations, andsurfactants. These excipients can contribute to the stability of thevirus. In some examples, the use of these excipients in the non-viralcompositions and thus in the viral formulations allows for the long-termstorage of viruses (e.g., storage for twelve months or greater) withoutloss of viral infectivity.

Suitable sugars for use in the non-viral compositions described hereininclude, for example, monosaccharides and disaccharides. In someexamples, the non-viral compositions include mannitol, sorbitol,sucrose, or combinations of these. Further examples of suitable sugarsinclude lactose, dextrose, fructose, glucose, and maltose. Optionally,the non-viral compositions and/or lyophilized viral formulations aresubstantially free of trehalose. Substantially free means that thenon-viral compositions and/or lyophilized viral formulations can includeless than 0.1%, less than 0.01%, less than 0.001%, less than 0.0001%, or0% of trehalose based on the weight of the formulation. In someexamples, the non-viral compositions and/or lyophilized viralformulations are substantially free of sugars other than sucrose (i.e.,the non-viral compositions and/or lyophilized viral formulation issubstantially free of non-sucrose polyols).

The sugars for use in the non-viral compositions and/or lyophilizedviral formulations can include one sugar or a combination of two or moresugars. For example, the non-viral composition can include sucrose asthe sugar present in the formulation or can include a combination ofmannitol and sorbitol as the sugars present in the formulation. Theconcentration of excipients, including sugars, present in thelyophilized viral formulations can be expressed herein as the weightpercent based on the weight of the liquid non-viral composition (i.e.,the non-viral composition, prior to lyophilization, including a liquidcarrier). The total concentration of sugar(s) present in the non-viralcomposition can be 10% by weight or less based on the weight of thenon-viral composition. For example, the total concentration of sugarscan be less than 7.5% by weight based on the weight of the liquidnon-viral composition (e.g., less than 7.4% by weight, less than 7.3% byweight, less than 7.2% by weight, less than 7.1% by weight, less than 7%by weight, less than 6% by weight, less than 5% by weight, less than 4%by weight, less than 3% by weight, less than 2% by weight, or less than1% by weight based on the weight of the liquid non-viral composition).For example, sucrose can be present in the non-viral composition in aconcentration ranging from 0.1% to 5%, from 1% to 4.5%, or from 2% to 4%(e.g., 3%) by weight based on the weight of the liquid non-viralcomposition. Optionally, mannitol and sorbitol can be included in thenon-viral composition in a combined concentration of less than 7.5%(e.g., 7%) based on the weight of the liquid non-viral composition. Forinstance, mannitol can be included in a concentration ranging from 0.01%to 7.4% (e.g., from 0.1% to 7%, from 1% to 6%, from 2% to 5%, or from 3%to 4%) and sorbitol can be included in a concentration ranging from0.01% to 7.4% (e.g., from 0.1% to 7%, from 1% to 6%, from 2% to 5%, orfrom 3% to 4%), such that the combined concentration of the sugars isless than 7.5% based on the weight of the liquid non-viral composition.

Amino acids can also be included in the non-viral compositions describedherein. Suitable amino acids include, for example, histidine, arginine,lysine, methionine, glutamic acid, or mixtures of these. One or moreamino acids can be present in the non-viral composition in aconcentration of 5% or less based on the weight of the liquid non-viralcomposition. For example, the concentration of amino acids can be 4.5%or less, 4.0% or less, 3.5% or less, 3.0% or less, 2.5% or less, 2.0% orless, 1.5% or less, 1.0% or less, or 0.5% or less based on the weight ofthe liquid non-viral composition.

Divalent cations can also be included in the non-viral compositionsdescribed herein. A suitable divalent cation for use in the non-viralcomposition includes the magnesium cation (i.e., Mg²⁺). Mg²⁺ can beintroduced to the non-viral composition in combination with an anion asa salt, such as MgCl₂. Optionally, the non-viral compositions and/orviral formulations are substantially free of Zn²⁺. The divalent cationcan be present in the liquid non-viral composition in a concentrationranging from 0.01 mM to 5 mM. For example, Mg²⁺ can be present in theviral formulation as MgCl₂ in a concentration ranging from 0.1 mM to 4.5mM, 0.5 mM to 4 mM, or 1 mM to 3 mM (e.g., 2 mM). Optionally, theexcipients present in the non-viral formulation, excluding the liquidcarrier, can be substantially free of monovalent cationic salts, suchas, for example, sodium (Na⁺), lithium (Li⁺), potassium (K⁺), andammonium (NH₄ ⁺) containing salts.

A further excipient for use in the non-viral compositions describedherein can include, for example, a surfactant. A surfactant refers to asubstance having, in combination, a hydrophilic moiety and a hydrophobicmoiety. As used herein, surfactants include, for example, detergents.Suitable surfactants for use in the non-viral compositions describedherein include ionic and non-ionic surfactants. In some examples,polysorbate 80 is optionally included as the non-ionic surfactant in thenon-viral compositions. One or more surfactants can be present in thenon-viral compositions, optionally in an amount of less than 1% byweight based on the weight of the liquid non-viral composition. Forexample, the surfactant(s) can be present in the non-viral compositionin an amount of less than 0.5% by weight, less than 0.1% by weight, orless than 0.05% by weight (e.g., 0.01% by weight).

Optionally, the non-viral compositions and/or lyophilized viralformulations are substantially free of carboxylates. Examples ofcarboxylates include succinate and citrate.

An exemplary combination of the virus and non-viral composition(including excipients) to form a viral formulation includes a purifiedvirus, mannitol, sorbitol, histidine, and Mg²⁺. As described above, thenon-viral composition, prior to lyophilization, can further include(i.e., in addition to the excipients) a liquid carrier to form a liquidnon-viral composition. The combined concentration of the sugars in theliquid non-viral composition can be less than 7.5% by weight based onthe weight of the liquid non-viral composition. For example, theconcentration of mannitol can be 3% and the concentration of histidinecan be 2% to provide a combined concentration of 5%. Optionally, thesorbitol can be present in a concentration of less than 3% based on theweight of the liquid non-viral composition. For example, sorbitol can bepresent in a concentration of less than 2.9%, less than 2.8%, less than2.7%, less than 2.6%, less than 2.5%, less than 2.4%, less than 2.3%,less than 2.2%, less than 2.1%, less than 2%, less than 1.9%, less than1.8%, less than 1.7%, less than 1.6%, less than 1.5%, less than 1.4%,less than 1.3%, less than 1.2%, less than 1.1%, or less than 1%. Theformulation can also include a non-ionic surfactant, such as polysorbate80, in an amount less than 0.1% by weight of the liquid non-viralcomposition (e.g., 0.01%). Further, the non-viral composition and/or theviral formulation can be substantially free of monovalent cationicsalts, Zn²⁺, and/or trehalose.

Another suitable viral formulation includes a purified virus and anon-viral composition including sucrose, Mg²⁺, and a non-ionicsurfactant. Prior to lyophilization the non-viral composition canfurther include a liquid carrier, thus forming a liquid non-viralcomposition. The concentration of sucrose in the liquid non-viralcomposition can be less than 5% based on the weight of the liquidnon-viral composition. Optionally, sucrose is present in a concentrationof 4.5% or less, 4% or less, 3.5% or less, 3% or less, 2.5% or less, or2% or less based on the weight of the liquid non-viral composition.Furthermore, the non-viral composition and/or the viral formulation canbe substantially free of non-sucrose polyols and carboxylates (e.g.,succinate and citrate).

Also described herein are methods of making the lyophilized viralformulations. The methods include providing a virus (e.g., a purifiedvirus), combining the virus with a non-viral composition including theexcipients in a liquid carrier to form a liquid viral formulation, andlyophilizing the liquid viral formulation. In some examples, a suitableamount of virus is provided to prepare a viral formulation at a titerranging from 1×10⁵ to 4×10¹² viral particles per milliliter (VP/mL) ofliquid carrier.

Suitable liquid carriers can be aqueous or non-aqueous carriers.Examples of suitable non-aqueous carriers include propylene glycol,polyethylene glycol, and oils, including those of petroleum, animal,vegetable or synthetic origin, such as peanut oil, soybean oil, mineraloil, sesame oil, olive oil, and the like. Organic esters such as ethyloleate are also suitable non-aqueous carriers. Aqueous carriers includewater, ethanol, glycerol, alcoholic/aqueous solutions, emulsions, orsuspensions, including saline and buffered media. Water or an aqueouscarrier is preferred when the pharmaceutical composition is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid carriers, particularly forinjectable solutions. The composition, if desired, can also containwetting or emulsifying agents, lubricants, glidants, emollients,humectants, thickeners, flavoring agents, preservatives, or pH buffers.pH buffers can be included to control the pH of the non-viralcomposition and thus, the viral formulation. In some examples, thebuffer is included to maintain the pH of the viral formulation between 5and 8.5. For example, the buffer can be included to maintain the pH ofthe viral formulation between 6.8 and 8.0 or between 7.0 and 7.8 (e.g.,7.4). Examples of suitable buffers include phosphate buffers such asphosphate buffered saline (PBS), e.g., 0.01-0.1 M and preferably 0.05 Mphosphate buffer, acetate buffers, benzoate buffers, citrate buffers,lactate buffers, maleate buffers, and tartrate buffers. Bufferedcarriers like Hanks's solution, Ringer's solution, dextrose solution, 5%human serum albumin, Ringer's dextrose, dextrose and sodium chloride,lactated Ringer's and fixed oils, polyethylene glycol, polyvinylpyrrolidone, or lecithin can be used. Monoethanolamine, diethanolamine,tromethamine, and glycine solutions can also be used as suitablebuffers. Liposomes and nonaqueous vehicles such as fixed oils may alsobe used as carriers. Further examples of suitable carriers are describedin “Remington's Pharmaceutical Sciences” by E. W. Martin. Theformulation should suit the mode of administration.

Alternatively, the non-viral composition containing the excipients in aliquid carrier can be added to a culture of cells infected with virus.As used herein, a culture of cells refers to a population of culturedcells as found in their culture conditions (e.g., the cells infectedwith virus and the culture medium).

Lyophilization can be performed using techniques and equipment as knownin the art. The lyophilization process can be performed, for example,using a lyophilizer. Lyophilizing can involve freezing and subsequentlydrying the liquid viral formation. Optionally the lyophilizationinvolves a product loading stage, freezing stage and primary drying andsecondary drying stage. The product is loaded into the lyophilizer andthe shelves are set to a target temperature setpoint for apre-determined duration. The freezing stage involved the shelves beingchilled to a target set point at a controlled rate (° C./hr). Theproduct is maintained at the freezing stage for a pre-determined amountof time. In the freezing step, the liquid viral formulation can becooled, for an appropriate period of time, to a temperature lower than0° C. to form a frozen viral formulation. Optionally, the liquid viralformulation can be cooled to a temperature of −50° C. or lower. In someexamples, the liquid viral formulation can be cooled for 10 hours orless. For example, the viral formulation can be cooled for 9 hours orless, 8 hours or less, 7 hours or less, 6 hours or less, 5 hours orless, 4 hours or less, 3 hours or less, 2 hours or less, 1 hour or less,or 30 minutes or less. Optionally, the lyophilization process caninclude an annealing step wherein the frozen viral formulation is warmedto a temperature at or below ambient temperature, and then cooled againto form a frozen viral formulation. In some examples, the annealing stepis not performed. The frozen viral formulation can then be dried underreduced pressure (e.g., by applying a vacuum) to form the lyophilizedviral formulation. Optionally, a vacuum pressure ranging from 50 to 80μm Hg (e.g., 60 μm Hg) can be applied to the frozen viral formulation.The drying step can be performed at a temperature at, below, or aboveambient temperature. For example, the drying step can be performed at atemperature of 40° C. or less, 30° C. or less, 20° C. or less, 10° C. orless, or 0° C. or less. Optionally, the lyophilized viral formulationcan be further dried in one or more additional drying steps at atemperature at, below, or above ambient temperature to remove residualwater. For example, the additional drying steps can be performed at atemperature ranging from −10° C. to 50° C. (e.g., from 0° C. to 40° C.,from 10° C. to 30° C., or from 20° C. to 25° C.). Furthermore, thelyophilized viral formulation can be dried in the presence of an inertgas (e.g., nitrogen) or a combination of inert gasses. For example, thelyophilization vessel and/or the viral storage container can be purgedwith an inert gas and capped to avoid exposure of the viral formation tothe air. The lyophilized viral formulation, after one or more dryingsteps, can have a moisture content of, for example, less than 20%. Insome examples, the moisture content of the lyophilized viral formulationis less than 15%, less than 10%, less than 5%, less than 4%, less than3%, less than 2%, less than 1%, less than 0.5%, or less than 0.1%.

The lyophilized viral formulations described herein can be used topreserve and stabilize a virus for a period of time, including extendedstorage periods. For example, the virus prepared according to theformulations described herein can be stored for up to twelve months(e.g., one day, one week, one month, three months, six months, ninemonths, or twelve months) without losing viral infectivity. Thelyophilized viral formulations described herein are stable at aboutambient temperature and may exhibit increased stability at temperatureslower than about ambient temperature. As used herein, ambienttemperature refers to a temperature between about 10° C. and about 30°C. Viruses can be stored in the viral formulations at temperatures belowambient temperature without significant loss of infectivity orimmunogenicity. In some examples, the lyophilized viral formulations arestored at temperatures of 9° C. and lower (e.g., 8° C. and lower, 7° C.and lower, 6° C. and lower, 5° C. and lower, 4° C. and lower, 3° C. andlower, 2° C. and lower, and 1° C. and lower. For example, the storagetemperature can range from 2° C. to 8° C. (e.g., 4° C.). Further, thelyophilized viral formulations can be stored at temperatures below 0°C., such as, for example, −20° C. or from −60° C. to −80° C., whilemaintaining viral infectivity.

The lyophilized viral formulations are stable, for example, at atemperature at about ambient temperature for a period of time (e.g., atleast one day). In some examples, the viral formulations are stable at atemperature of about 4° C. or lower for at least three months (e.g., atleast four months, at least five months, at least six months, at leastseven months, at least eight months, at least nine months, at least tenmonths, at least eleven months, at least twelve months, at leastthirteen months, at least fourteen months, at least fifteen months, atleast sixteen months, at least seventeen months, at least eighteenmonths, or any amount of time greater than three months). Additionallydescribed herein are methods of preparing viral formulations with lowlevels of particulates, e.g., low or non-aggregating viral formulations.The methods include preparing the viral formulation as described hereinand then lyophilizing the formulation to prepare a lyophilized viralformulation. The viral formulations prepared according to these methodsinclude low levels of particulates and are thus suitable foradministration by parenteral infusion or injection. In some examples,the levels of particulates in the methods are determined using the lightobscuration particle count test and/or the microscopic particle counttest according to USP <788>, which is incorporated herein in itsentirety. Optionally, the viral formulations comprise fewer than 6,000particles having a particle size of 10 microns or greater per container.For example, the viral formulations can comprise fewer than 5,000particles, fewer than 4,000 particles, fewer than 3,000 particles, fewerthan 2,000 particles, fewer than 1,000 particles, fewer than 900particles, fewer than 800 particles, fewer than 700 particles, fewerthan 600 particles, fewer than 500 particles, fewer than 400 particles,fewer than 300 particles, fewer than 200 particles, or fewer than 100particles of 10 microns or greater per container. Optionally, the viralformulations comprise fewer than 600 particles having a particle size of25 microns or greater (e.g., fewer than 500 particles, fewer than 400particles, fewer than 300 particles, fewer than 200 particles, fewerthan 100 particles, fewer than 50 particles, or fewer than 10 particlesof 25 microns or greater per container).

Further provided herein are pharmaceutical compositions including thelyophilized viral formulations. The herein provided compositions can beadministered in vitro or in vivo in a pharmaceutically acceptablecarrier. Optionally, the lyophilized viral formulations can bereconstituted by dissolving or suspending the lyophilized viralformulation in a medium prior to combining with a pharmaceuticallyacceptable carrier. A pharmaceutically acceptable carrier can be asolid, semi-solid, or liquid material that can act as a vehicle,carrier, or medium for the lyophilized viral formulation. Thus, thelyophilized viral formulation can be in the form of tablets, soft orhard gelatin capsules, suspensions, emulsions, solutions, syrups,aerosols (in a liquid medium), and sterile injectable solutions.Optionally, the lyophilized viral formulations are suitable forinfusion. In these examples, the lyophilized viral formulations preparedaccording to the methods described herein can be reconstituted andfurther diluted, as appropriate, for infusion. For example, thenon-aggregating viral formulations having lower levels of particulatesas described herein can be reconstituted and the reconstituted viralformulations can be suitable for parenteral infusion or injection.

A pharmaceutical composition additionally can include, withoutlimitation, lubricating agents such as talc, magnesium stearate, andmineral oil; wetting agents; emulsifying and suspending agents;preserving agents such as methyl- and propylhydroxy-benzoates;sweetening agents; and flavoring agents. Pharmaceutical compositions canbe formulated to provide quick, sustained, or delayed release of thevirus included in the lyophilized viral formulation after administrationby employing procedures known in the art. In addition to therepresentative formulations described below, other suitable formulationsfor use in a pharmaceutical composition can be found in Remington: TheScience and Practice of Pharmacy (21th ed.) ed. David B. Troy,Lippincott Williams & Wilkins, 2005.

Liquid formulations that include the lyophilized viral formulations(e.g., reconstituted lyophilized viral formulations) for oraladministration or for injection generally include aqueous solutions,suitably flavored syrups, aqueous or oil suspensions, and flavoredemulsions with edible oils such as corn oil, cottonseed oil, sesame oil,coconut oil, or peanut oil, as well as elixirs and similarpharmaceutical vehicles.

Another formulation that is optionally employed in the methods of thepresent disclosure includes transdermal delivery devices (e.g.,patches). Such transdermal delivery can be used to provide continuous ordiscontinuous infusion of the virus included in the lyophilized viralformulations as described herein. The construction and use oftransdermal patches for the delivery of pharmaceutical agents is wellknown in the art. See, for example, U.S. Pat. No. 5,023,252. Suchpatches can be constructed for continuous, pulsatile, or on-demanddelivery of the viruses.

In the provided methods, the lyophilized viral formulation isadministered in a manner so that the virus can ultimately contact thetarget cells, for example, systemically. The route by which the viralformulation is administered depends on the location as well as the typeof the target cells. A wide variety of administration routes can beemployed. For vaccines, the virus can be administered systemically,intradermally, or subcutaneously, so as to target antigen presentingcells, so as to elicit an immune response. For oncolytic viruses wherethe target is an accessible solid tumor, the viral formulation can beadministered by injection directly to the tumor or intravenously.Optionally, the formulation is suitable for reconstitution prior toadministration. For a hematopoietic tumor, for example, the viralformulation can be reconstituted and administered intravenously orintravascularly. For tumors that are not easily accessible within thebody, such as metastases, the viral formulation can be administered in amanner such that it can be transported systemically through the body ofthe mammal and thereby reach the tumor (e.g., intravenously orintramuscularly). Alternatively, the viral formulation can beadministered directly to a single solid tumor, where it then is carriedsystemically through the body to metastases. For vaccine or oncolytictherapy, the viral formulation can also be administered subcutaneously,intraperitoneally, intrathecally (e.g., for brain tumor), topically(e.g., for melanoma), orally (e.g., for oral or esophageal cancer),rectally (e.g., for colorectal cancer), vaginally (e.g., for cervical orvaginal cancer), nasally, by inhalation spray or by aerosol formulation(e.g., for lung cancer).

Optionally, the viral formulation is administered continuously to asubject at least once per day or up to throughout the day on consecutivedays, for a period of time. For vaccine administration, typically aprime and one or more boost administrations are necessary. Thus, theviral formulation is administered, for example, to a subject in anypharmacologically acceptable solution (e.g., by intravenousadministration or infusion) over a period of time or intermittently. Forexample, the formulation may be administered systemically by injection(e.g., IM or subcutaneously) or taken orally daily at least once perday, or administered by infusion in a manner that results in the dailydelivery into the tissue or blood stream of the subject. When the viralformulation is administered by infusion over a period of time, theperiod of time is, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, or 24hours, or any time between 1 and 24 hours, inclusive, or more.Optionally, the period of time is 5, 15, 30, 60, 90, 120, 150 or 180minutes, or any time between 5 and 180 minutes, inclusive, or more.Thus, for example, the viral formulation is administered by infusion for60 minutes. Administrations can be repeated daily for 2, 3, 4, 5, 6, 7,8, 9, 10, 14, 21, 28 days, or any number of days between 2 and 28 days,inclusive, or longer.

Optionally, the viral formulation includes an adjuvant when used toelicit an immune response. Adjuvants include, for example, aluminumsalts, mineral oil, particulates, lipopolysaccharides, saponins, and thelike.

The viral formulations as disclosed herein can be administered in anamount that is sufficient (i.e., an effective amount) to treat aproliferative disorder or to elicit an immune response. A proliferativedisorder is treated when administration of the liquid virus formulationto proliferating cells affects lysis (e.g., oncolysis) of the affectedcells, resulting in a reduction in the number of abnormally,proliferating cells, a reduction in the size of a neoplasm, and/or areduction in or elimination of symptoms (e.g., pain) associated with theproliferating disorder. As used herein, the term oncolysis means atleast 10% of the proliferating cells are lysed (e.g., at least about20%, 30%, 40%, 50%, or 75% of the cells are lysed). The percentage oflysis can be determined, for example, by measuring the reduction in thesize of a neoplasm or in the number of proliferating cells in a mammal,or by measuring the amount of lysis of cells in vitro (e.g., from abiopsy of the proliferating cells).

An effective amount of the viral formulation will be determined on anindividual basis and may be based, at least in part, on the particularvirus used in the viral formulation; the individual's size, age, gender;the goal of the treatment (e.g., to treat a proliferative disease or toelicit an immune response); and the size and other characteristics ofthe abnormally proliferating target cells. The viral concentration inthe viral formulation can be measured by determining the number ofplaque forming units (PFU) in plaque based assays. For example, fortreatment of a human with a proliferative disease, approximately 10³ to10¹² PFU of a virus contained in a viral formulation are used, dependingon the type, size, and number of proliferating cells or neoplasmspresent. The effective amount can be, for example, from about 1.0 PFU/kgbody weight to about 10¹⁵ PFU/kg body weight (e.g., from about 10²PFU/kg body weight to about 10¹³ PFU/kg body weight). For example, theeffective amount of the viral formulation administered daily can be1×10¹⁰ PFU and the viral formulation can be administered over five daysto result in a total treatment amount of 5×10¹⁰. Optionally, the viralconcentration in the viral formulation can be measured by determiningthe 50% tissue culture infective dose (TCID₅₀), which is the amount ofvirus required to produce a cytopathic effect in 50% of cells infectedwith the virus. In some examples, the ratio between TCID₅₀ and PFU is3:1. The effective amount of the viral formulation for treating a humanwith a proliferative disease can be from about 1×10⁸ to about 1×10¹⁵TCID₅₀ per day. The effective amount of the viral formulation can beadministered over a period of time, referred to herein as a cycle. Acycle can represent, for example, one day, two days, three days, fourdays, five days, six days, seven days, eight days, nine days, or tendays, where an equal or different amount of the viral formulation can beadministered daily. For example, 3×10¹⁰ TCID₅₀ per day can beadministered over five days to result in a total amount of 1.5×10¹¹TCID₅₀ per cycle. Optionally, the effective amount is about 3×10¹⁰TCID₅₀ per day. Optionally, the viral formulation is administered as aone hour intravenous infusion.

Optimal dosages of the viral formulations depend on a variety offactors. The exact amount required will vary from subject to subject,depending on the species, age, weight and general condition of thesubject, the severity of the disease being treated, the particular virusused in the formulation, and its mode of administration. Thus, it is notpossible to specify an exact amount for every formulation. However, anappropriate amount can be determined by one of ordinary skill in the artusing only routine experimentation given the guidance provided herein.

The dosage ranges for the administration of the formulations are thoselarge enough to produce the desired effect in which the symptoms of thedisease are affected or large enough to elicit an immune response. Thedosage should not be so large as to cause adverse side effects, such asunwanted cross-reactions and anaphylactic reactions. The dosage can beadjusted by the individual physician in the event of anycontraindications.

Dosages vary and are administered in one or more dose administrationsdaily, for one or several days. The provided viral formulation can beadministered in a single dose or in multiple doses (e.g., two, three,four, six, or more doses). For example, where the administration is byinfusion, the infusion can be a single sustained dose or can bedelivered by multiple infusions. Treatment may last from several days toseveral months or until diminution of the disease is achieved.

Combinations of the viral formulations can be administered eitherconcomitantly (e.g., as an admixture), separately but simultaneously(e.g., via separate intravenous lines into the same subject), orsequentially (e.g., one of the formulations is given first followed bythe second). Thus, the term combination is used to refer to eitherconcomitant, simultaneous, or sequential administration of two or moreagents.

It is contemplated that the provided viral formulations may be combinedwith other tumor therapies such as chemotherapy, radiotherapy, surgery,hormone therapy, and/or immunotherapy. Thus, the viral formulations maybe administered in conjunction with surgery or removal of the neoplasm.Therefore, provided herewith are methods for the treatment of a solidneoplasm comprising surgical removal of the neoplasm and administrationof the viral formulation at or near to the site of the neoplasm.

It is further contemplated that the viral formulations in the providedmethods are, optionally, administered in combination with or in additionto known anticancer compounds or chemotherapeutic agents.Chemotherapeutic agents are compounds which may inhibit the growth oftumors. Such agents, include, but are not limited to, antineoplasticagents such as Acivicin; Aclarubicin; Acodazole Hydrochloride; AcrQnine;Adozelesin; Aldesleukin; Altretamine; Ambomycin; Ametantrone Acetate;Aminoglutethimide; Amsacrine; Anastrozole; Anthramycin; Asparaginase;Asperlin; Azacitidine; Azetepa; Azotomycin; Batimastat; Benzodepa;Bicalutamide; Bisantrene Hydrochloride; Bisnafide Dimesylate; Bizelesin;Bleomycin Sulfate; Brequinar Sodium; Bropirimine; Busulfan;Cactinomycin; Calusterone; Caracemide; Carbetimer; Carboplatin;Carmustine; Carubicin Hydrochloride; Carzelesin; Cedefingol;Chlorambucil; Cirolemycin; Cisplatin; Cladribine; Crisnatol Mesylate;Cyclophosphamide; Cytarabine; Dacarbazine; Dactinomycin; DaunorubicinHydrochloride; Decitabine; Dexormaplatin; Dezaguanine; DezaguanineMesylate; Diaziquone; Docetaxel; Doxorubicin; Doxorubicin Hydrochloride;Droloxifene; Droloxifene Citrate; Dromostanolone Propionate; Duazomycin;Edatrexate; Eflomithine Hydrochloride; Elsamitrucin; Enloplatin;Enpromate; Epipropidine; Epirubicin; Epirubicin Hydrochloride;Erbulozole; Esorubicin Hydrochloride; Estramustine; EstramustinePhosphate Sodium; Etanidazole; Ethiodized Oil I 131; Etoposide;Etoposide Phosphate; Etoprine; Fadrozole Hydrochloride; Fazarabine;Fenretinide; Floxuridine; Fludarabine Phosphate; 5-Fluorouracil;Flurocitabine; Fosquidone; Fostriecin Sodium; Gemcitabine; GemcitabineHydrochloride; Gold Au 198; Hydroxyurea; Idarubicin Hydrochloride;Ifosfamide; Ilmofosine; Interferon Alfa-2a; Interferon Alfa-2b;Interferon Alfa-n1; Interferon Alfa-n3; Interferon Beta-I a; InterferonGamma-I b; Iproplatin; Irinotecan Hydrochloride; Lanreotide Acetate;Letrozole; Leuprolide Acetate; Liarozole Hydrochloride; LometrexolSodium; Lomustine; Losoxantrone Hydrochloride; Masoprocol; Maytansine;Mechlorethamine Hydrochloride; Megestrol Acetate; Melengestrol Acetate;Melphalan; Menogaril; Mercaptopurine; Methotrexate; Methotrexate Sodium;Metoprine; Meturedepa; Mitindomide; Mitocarcin; Mitocromin; Mitogillin;Mitomalcin; Mitomycin C; Mitosper; Mitotane; Mitoxantrone; MitoxantroneHydrochloride; Mycophenolic Acid; Nocodazole; Nogalamycin; Ormaplatin;Oxisuran; Paclitaxel; Pegaspargase; Peliomycin; Pentamustine; PeplomycinSulfate; Perfosfamide; Pipobroman; Piposulfan; PiroxantroneHydrochloride; Plicamycin; Plomestane; Porfimer Sodium; Porfiromycin;Prednimustine; Procarbazine Hydrochloride; Puromycin; PuromycinHydrochloride; Pyrazofurin; Riboprine; Rogletimide; Safmgol; SafingolHydrochloride; Semustine; Simtrazene; Sparfosate Sodium; Sparsomycin;Spirogermanium Hydrochloride; Spiromustine; Spiroplatin; Streptonigrin;Streptozocin; Strontium Chloride Sr 89; Sulofenur; Talisomycin; Taxane;Taxoid; Tecogalan Sodium; Tegafur; Teloxantrone Hydrochloride;Temoporfin; Teniposide; Teroxirone; Testolactone; Thiamiprine;Thioguanine; Thiotepa; Tiazofurin; Tirapazamine; TopotecanHydrochloride; Toremifene Citrate; Trestolone Acetate; TriciribinePhosphate; Trimetrexate; Trimetrexate Glucuronate; Triptorelin;Tubulozole Hydrochloride; Uracil Mustard; Uredepa; Vapreotide;Verteporfin; Vinblastine Sulfate; Vincristine Sulfate; Vindesine;Vindesine Sulfate; Vinepidine Sulfate; Vinglycinate Sulfate;Vinleurosine Sulfate; Vinorelbine Tartrate; Vinrosidine Sulfate;Vinzolidine Sulfate; Vorozole; Zeniplatin; Zinostatin; ZorubicinHydrochloride.

Further examples of anti-neoplastic compounds include 20-epi-1,25dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin;acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TK antagonists;altretamine; ambamustine; amidox; amifostine; aminolevulinic acid;amrubicin; atrsacrine; anagrelide; anastrozole; andrographolide;angiogenesis inhibitors; antagonist D; antagonist G; antarelix;anthracyclines; anti-dorsalizing morphogenetic protein-1; antiandrogens,prostatic carcinoma; antiestrogens; antineoplastons; antisenseoligonucleotides; aphidicolin glycinate; apoptosis gene modulators;apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; argininedeaminase; aromatase inhibitors; asulacrine; atamestane; atrimustine;axinastatin 1; axinastatin 2; axinastatin 3; azasetron; azatoxin;azatyrosine; baccatin III derivatives; balanol; batimastat; BCR/ABLantagonists; benzochlorins; benzoylstaurosporine; beta lactamderivatives; beta-alethine; betaclamycin B; betulinic acid; bFGFinhibitor; bicalutamide; bisantrene; bisaziridinylspermine; bisnafide;bistratene A; bizelesin; breflate; bropirimine; budotitane; buthioninesulfoximine; calcipotriol; calphostin C; camptothecin derivatives;canarypox IL-2; capecitabine; carboxamide-amino-triazole;carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor;carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropinB; cetrorelix; chlorins; chloroquinoxaline sulfonamide; cicaprost;cis-porphyrin; cladribine; clomifene analogues; clotrimazole;collismycin A; collismycin B; combretastatin A4; combretastatinanalogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8;cryptophycin A derivatives; curacin A; cyclopentanthraquinones;cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor;cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin;dexifosfamide; dexrazoxane; dexverapamil; diaziquone; didemnin B; didox;diethylnorspermine; dihydro-5-azacytidine; dihydrotaxol, 9-; dioxamycin;diphenyl spiromustine; docosanol; dolasetron; doxifluridine;droloxifene; dronabinol; duocannycin SA; ebselen; ecomustine;edelfosine; edrecolomab; eflornithine; elemene; emitefur; epirubicin;epristeride; estramustine analogue; estrogen agonists; estrogenantagonists; etanidazole; etoposide phosphate; exemestane; fadrozole;fazarabine; fenretinide; filgrastim; fmasteride; flavopiridol;flezelastine; fluasterone; fludarabine; fluorodaunorunicinhydrochloride; forfenimex; formestane; fostriecin; fotemustine;gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix;gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam;heregulin; hexamethylene bisacetamide; hormone therapies; hypericin;ibandronic acid; idarubicin; idoxifene; idramantone; ilmofosine;ilomastat; imidazoacridones; imiquimod; immunostimulant peptides;insulin-like growth factor-1 receptor inhibitor; interferon agonists;interferons; interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-;irinotecan; iroplact; irsogladine; isobengazole; isohomohalicondrin B;itasetron; jasplakinolide; kahalalide F; lamellarin-N triacetate;lanreotide; leinamycin; lenograstim; lentinan sulfate; leptolstatin;letrozole; leukemia inhibiting factor; leukocyte alpha interferon;leuprolide+estrogen+progesterone; leuprorelin; levamisole; LHRH analogs;liarozole; linear polyamine analogue; lipophilic disaccharide peptide;lipophilic platinum compounds; lissoclinamide 7; lobaplatin; lombricine;lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine;lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides;maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysininhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone;meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone;miltefosine; mirimostim; mismatched double stranded RNA; mitoguazone;mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growthfactor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonalantibody, human chorionic gonadotrophin; monophosphoryl lipidA+myobacterium cell wall sk; mopidamol; multiple drug resistance genieinhibitor; multiple tumor suppressor 1-based therapy; mustard anticanceragent; mycaperoxide B; mycobacterial cell wall extract; myriaporone;N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip;naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin;nemorubicin; neridronic acid; neutral endopeptidase; nilutamide;nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn;O6-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone;ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin;osaterone; oxaliplatin; oxaunomycin; paclitaxel analogues; paclitaxelderivatives; palauamine; palmitoylrhizoxin; pamidronic acid;panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase;peldesine; pentosan polysulfate sodium; pentostatin; pentrozole;perflubron; perfosfamide; perillyl alcohol; phenazinomycin;phenylacetate; phosphatase inhibitors; picibanil; pilocarpinehydrochloride; pirarubicin; piritrexim; placetin A; placetin B;plasminogen activator inhibitor; platinum complex; platinum compounds;platinum-triamine complex; porfimer sodium; porfiromycin; prednisone;progestational agents; propyl bis-acridone; prostaglandin J2; proteasomeinhibitors; protein A-based immune modulator; protein kinase Cinhibitor; protein kinase C inhibitors, microalgal; protein tyrosinephosphatase inhibitors; purine nucleoside phosphorylase inhibitors;purpurins; pyrazoloacridine; pyridoxylated hemoglobin polyoxyethyleneconjugate; raf antagonists; raltitrexed; ramosetron; ras farnesylprotein transferase inhibitors; ras inhibitors; ras-GAP inhibitor;retelliptine demethylated; rhenium Re 186 etidronate; rhizoxin;ribozymes; RII retinamide; rogletimide; rohitukine; romurtide;roquinimex; rubiginone B1; ruboxyl; safingol; saintopin; SarCNU;sarcophytol A; sargramostim; Sdi 1 mimetics; semustine; senescencederived inhibitor 1; sense oligonucleotides; signal transductioninhibitors; signal transduction modulators; single chain antigen bindingprotein; sizofiran; sobuzoxane; sodium borocaptate; sodiumphenylacetate; solverol; somatomedin binding protein; sonermin;sparfosic acid; spicamycin D; spiromustine; splenopentin; spongistatin1; squalamine; stem cell inhibitor; stem-cell division inhibitors;stipiamide; stromelysin inhibitors; sulfmosine; superactive vasoactiveintestinal peptide antagonist; suradista; suramin; swainsonine;synthetic glycosaminoglycans; tallimustine; tamoxifen; tamoxifenmethiodide; tauromustine; tazarotene; tecogalan sodium; tegafur;tellurapyrylium; telomerase inhibitors; temoporfin; temozolomide;teniposide; tetrachlorodecaoxide; tetrazomine; thaliblastine;thalidomide; thiocoraline; thrombopoietin; thrombopoietin mimetic;thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroidstimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocenedichloride; topotecan; topsentin; toremifene; totipotent stem cellfactor; translation inhibitors; tretinoin; triacetyluridine;triciribine; trimetrexate; triptorelin; tropisetron; turosteride;tyrosine kinase inhibitors; tyrphostins; UBC inhibitors; ubenimex;urogenital sinus-derived growth inhibitory factor; urokinase receptorantagonists; vapreotide; variolin B; vector system, erythrocyte genetherapy; velaresol; veramine; verdins; verteporfin; vinorelbine;vinxaltine; vitaxin; vorozole; zanoterone; zeniplatin; zilascorb;zinostatin stimalamer.

Further examples of chemotherapeutic agents suitable for use in theviral formulations described herein include anti-cancer supplementarypotentiating agents, including tricyclic anti-depressant drugs (e.g.,imipramine, desipramine, amitryptyline, clomiprainine, trimipramine,doxepin, nortriptyline, protriptyline, amoxapine and maprotiline);non-tricyclic anti-depressant drugs (e.g., sertraline, trazodone andcitalopram); agents that recognize or block VEGF (e.g., Avastin); Ca²⁺antagonists (e.g., verapamil, nifedipine, nitrendipine and caroverine);Calmodulin inhibitors (e.g., prenylamine, trifluoroperazine andclomipramine); Amphotericin B; Triparanol analogues (e.g., tamoxifen);antiarrhythmic drugs (e.g., quinidine); antihypertensive drugs (e.g.,reserpine); antibodies to receptors, such as herceptin; thiol depleters(e.g., buthionine and sulfoximine); and multiple drug resistancereducing agents such as Cremaphor EL. The viral formulations describedherein can also be administered with cytokines such as granulocytecolony stimulating factor.

As used herein, the term proliferative disorder refers to any cellulardisorder in which the cells proliferate more rapidly than normal tissuegrowth. A proliferative disorder includes, but is not limited to,neoplasms, which are also referred to as tumors. A neoplasm can include,but is not limited to, pancreatic cancer, breast cancer, brain cancer(e.g., glioblastoma), lung cancer, prostate cancer, colorectal cancer,thyroid cancer, renal cancer, adrenal cancer, liver cancer,neurofibromatosis 1, and leukemia. A neoplasm can be a solid neoplasm(e.g., sarcoma or carcinoma) or a cancerous growth affecting thehematopoietic system (e.g., lymphoma or leukemia). Other proliferativedisorders include, but are not limited to neurofibromatosis.

As used herein the terms treatment, treat, treating or amelioratingrefers to a method of reducing the effects of a disease or condition orsymptom of the disease or condition. Thus in the disclosed method,treatment can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or100% reduction or amelioration in the severity of an established diseaseor condition or symptom of the disease or condition. For example, themethod for treating cancer is considered to be a treatment if there is a10% reduction in one or more symptoms of the disease in a subject ascompared to control. Thus the reduction can be a 10, 20, 30, 40, 50, 60,70, 80, 90, 100%, or any percent reduction in between 10 and 100 ascompared to native or control levels. It is understood that treatmentdoes not necessarily refer to a cure or complete ablation of thedisease, condition, or symptoms of the disease or condition.

As used herein, the term subject can be a vertebrate, more specificallya mammal (e.g., a human, horse, pig, rabbit, dog, sheep, goat, non-humanprimate, cow, cat, guinea pig or rodent), a fish, a bird or a reptile oran amphibian. The term does not denote a particular age or sex. Thus,adult and newborn subjects, whether male or female, are intended to becovered. As used herein, patient or subject may be used interchangeablyand can refer to a subject with a disease or disorder. The term patientor subject includes human and veterinary subjects.

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application.

A number of aspects have been described. Nevertheless, it will beunderstood that various modifications may be made. Furthermore, when onecharacteristic or step is described it can be combined with any othercharacteristic or step herein even if the combination is not explicitlystated. Accordingly, other aspects are within the scope of the claims.

EXAMPLES Example 1 Non-Viral Composition Preparation

Compositions 1 and 2 were prepared by mixing the components shown inTable 1.

TABLE 1 Composition 1 Composition 2 Component Amount Component AmountSugar(s) Mannitol 30 mg/mL Sucrose 40 mg/mL Sorbitol 20 mg/mL AminoHistidine 20 mg/mL N/A N/A Acid Divalent MgCl₂ (2 mM) 2 mM MgCl₂ 2 mMCation Surfactant Polysorbate 80 0.01% Polysorbate 80 0.05% CarrierPhosphate Buffered Saline Phosphate Buffered Saline (PBS) (PBS)The PBS used contained 0.20 mg/mL potassium chloride, 8.00 mg/mL sodiumchloride, 0.24 mg/mL monobasic potassium phosphate, and 2.71 mg/mLdibasic sodium phosphate heptahydrate.

Example 2 Lyophilized Viral Formulation Preparation

A control reovirus formulation (Control Formulation) was prepared byproviding reovirus in phosphate buffered saline (PBS), further dilutingthe mixture with additional PBS to obtain a target total viral particletiter of 3×10¹⁰ TCID₅₀/mL, and lyophilizing the viral formulation.Formulation 1 was prepared by providing reovirus in PBS, diluting themixture with Composition 1 to obtain a target total viral particle titerof 3×10¹⁰ TCID₅₀/mL, and lyophilizing the viral formulation. Formulation2 was prepared by providing reovirus in PBS, diluting the mixture withComposition 2 to obtain a target total viral particle titer of 3×10¹⁰TCID₅₀/mL, and lyophilizing the viral formulation.

To lyophilize the viral formulations, each diluted composition wasindividually added to a lyophilization vessel and placed on a shelfwithin an ADVANTAGE PLUS EL-85 BENCHTOP FREEZE DRYER (The Virtis Co.,Inc; Gardiner, N.Y.). The shelves were set to a target temperaturesetpoint of 5° C. and compositions were maintained at this temperaturefor 120 minutes. The compositions were then cooled, at a rate of 15° C.per hour, to −50° C. and the composition was maintained at −50° C. forthree hours. The condenser of the lyophilizer was then cooled to atemperature between −75° C. and −50° C. and the chamber was evacuated toa target pressure of 60 μm Hg. The chamber pressure was controlled byadding 0.2 μm of filtered ambient air into the chamber. The compositionswere then warmed to −35° C. over 30 minutes and were dried at thistemperature for 48 hours. The temperature was then raised to 25° C. andthe compositions were dried for an additional 15 hours at thistemperature. The dilution ratios for each of the formulations weredetermined by analytical HPLC.

Example 3 Lyophilized Viral Formulation Storage

The infectious particle titers (TCID₅₀/mL) for Control Formulation,Formulation 1, and Formulation 2, as prepared in Example 2, weredetermined at ambient temperature after preparation (i.e., at time=0).At t=0, the Control Formulation was below the limit of detection,demonstrating that no viable virus was present. Thus, the ControlFormulation was not tested at any further timepoints. Formulations 1 and2 were stored for 0, 3, 6.5, 12 and 18.5 months at differenttemperatures, including 37° C., ambient temperature, temperaturesranging from 2° C. to 8° C., −20° C., and −80° C. After the storageperiods, the TCID₅₀ data for each of the formulations at the differenttemperatures were determined in triplicate. The mean data forFormulations 1 and 2 are shown in FIG. 1. The recoveries of infectiousparticle titers for Formulations 1 and 2 are shown in FIG. 2. The viraltiters were normalized to account for interassay variations in thecontrol titers. The normalized total viral titers, measured by HPLC, areshown in FIG. 3 for Formulation 1 and in FIG. 4 for Formulation 2.

The infectious particle titers of Formulation 1 stored for 3, 6.5, 12,and 18.5 months at −80° C., −20° C., and 2-8° C. were not significantlydifferent from the initial titers obtained at time=0 at ambienttemperature. However, in Formulation 1, drops in titer were observed atambient temperature after 3, 6.5, and 12 months. The infectious particletiters of Formulation 2 stored for 3, 6.5, 12, and 18.5 months at −80°C., −20° C., and 2-8° C. were stable or showed slight decreases ininfectivity as compared to the time=0 data. Similarly, slight decreasesin infectivity were observed in comparison with the time=0 data afterstorage for 3 and 6.5 months at ambient temperature. Formulation 2showed a decrease in infectivity after 12 months.

Example 4 Non-Aggregating Viral Formulations

Samples of Control Formulation and Composition 1, as described above,were tested, prior to lyophilization, for particulate matter using theUSP <788> Light Obscuration Particle Count Test. The results, as shownin Table 2, indicate that viral formulations prepared with Composition 1had many fewer particles than viral formulations using the ControlFormulation.

TABLE 2 Mean Particu- Particu- Par- late mat- late mat- ticle ter ≧10ter ≧25 Final Fill Bulk Formu- Diame- micron micron # Lot # Lot # lationter (nm) #/container #/container 1 122-08002 160-08001 Control 122.32349 32 2 122-08003 160-08001 Control 114.3 2490 53 3 160-10006160-10004 Comp. 1 121.8 50 4 4 160-10011 160-10008 Comp. 1 124.9 20 1

What is claimed is:
 1. A viral formulation, comprising: (a) a purifiedvirus; and (b) a non-viral composition comprising: (i) sucrose; (ii)Mg²⁺; and (iii) a non-ionic surfactant, wherein the viral formulation islyophilized; wherein the non-viral composition, prior to lyophilization,is a liquid non-viral composition further comprising a liquid carrier;wherein the concentration of sucrose in the liquid non-viralcomposition, prior to lyophilization, is less than 4.5% based on theweight of the liquid non-viral composition; wherein the liquid non-viralcomposition, excluding the liquid carrier, is free of non-sucrosepolyols, and carboxylates; wherein the non-viral composition is providedin an amount effective to stabilize the purified virus; and whereinmonovalent cationic salts are not present in the non-viral compositionas excipients contributing to the stability of the virus.
 2. The viralformulation of claim 1, wherein the non-viral composition furthercomprises a non-ionic surfactant.
 3. The viral formulation of claim 2,wherein the non-ionic surfactant is polysorbate
 80. 4. The viralformulation of claim 1, wherein the virus is an oncolytic virus, anon-enveloped virus, or a reovirus.
 5. The viral formulation of claim 4,wherein the reovirus is selected from the group consisting of amammalian reovirus, a recombinant or reassorted reovirus, and IDAC#190907-01.
 6. The viral formulation of claim 5, wherein the mammalianreovirus is a human reovirus.
 7. The viral formulation of claim 6,wherein the human reovirus is a serotype 3 reovirus.
 8. The viralformulation of claim 7, wherein the serotype 3 reovirus is a Dearingstrain serotype 3 reovirus.
 9. The viral formulation of claim 1, whereinMg²⁺ is present as magnesium chloride.
 10. The viral formulation ofclaim 1, wherein the liquid carrier is an aqueous carrier.
 11. The viralformulation of claim 1, wherein the viral formulation is stable at atemperature at about ambient temperature.
 12. The viral formulation ofclaim 1, wherein the viral formulation is stable at a temperature ofabout 4° C. or lower for at least three months, at least six months, atleast twelve months, or at least eighteen months.
 13. The viralformulation of claim 1, wherein the viral formulation is suitable forreconstitution before administration.
 14. A viral formulation,consisting essentially of: (a) a purified virus; and (b) a non-viralcomposition comprising: (i) sucrose; (ii) Mg²⁺; and (iii) a non-ionicsurfactant, wherein the viral formulation is lyophilized; wherein thenon-viral composition, prior to lyophilization, is a liquid non-viralcomposition further comprising a liquid carrier; wherein theconcentration of sucrose in the liquid non-viral composition, prior tolyophilization, is less than 4.5% based on the weight of the liquidnon-viral composition; wherein the non-viral composition is provided inan amount effective to stabilize the purified virus; and whereinmonovalent cationic salts are not present in the non-viral compositionas excipients contributing to the stability of the virus.
 15. A methodof making a viral formulation, comprising the steps of: (a) providing avirus; (b) combining, to form a liquid viral formulation, the virus anda liquid non-viral composition, wherein the liquid non-viral compositioncomprises: (i) sucrose in a concentration of less than 4.5% based on theweight of the liquid non-viral composition; Mg²⁺; (iii) a non-ionicsurfactant; and (iv) a liquid carrier, wherein the liquid non-viralcomposition, excluding the liquid carrier, is free of monovalentcationic salts, non-sucrose polyols, and carboxylates wherein thenon-viral composition is provided in an amount effective to stabilizethe virus; and wherein monovalent cationic salts are not present in thenon-viral composition as excipients contributing to the stability of thevirus; and (c) lyophilizing the liquid viral formulation, to form aviral formulation.
 16. The method of claim 15, further comprising addinga non-ionic surfactant to the liquid non-viral composition.
 17. Themethod of claim 16, wherein the non-ionic surfactant is polysorbate 80.18. The method of claim 15, wherein lyophilizing the liquid viralformulation comprises: (a) freezing the liquid viral formulation to atemperature lower than 0° C. to form a frozen viral formulation; and (b)applying a vacuum to the frozen viral formulation.
 19. The method ofclaim 15, further comprising reconstituting the lyophilized viralformulation.
 20. The method of claim 19, wherein reconstituting thelyophilized viral formulation comprises dissolving or suspending thelyophilized viral formulation in a medium.
 21. The method of claim 15,wherein the virus is an oncolytic virus, a non-enveloped virus, or areovirus.
 22. The method of claim 21, wherein the reovirus is selectedfrom the group consisting of a mammalian reovirus, a recombinant orreassorted reovirus, and IDAC #190907-01.
 23. The method of claim 22,wherein the mammalian reovirus is a human reovirus.
 24. The method ofclaim 23, wherein the human reovirus is a serotype 3 reovirus.
 25. Themethod of claim 24, wherein the serotype 3 reovirus is a Dearing strainserotype 3 reovirus.
 26. The method of claim 15, wherein Mg²⁺ is presentas magnesium chloride.
 27. The method of claim 15, wherein the liquidcarrier is an aqueous carrier.
 28. A viral formulation preparedaccording to the method of claim
 15. 29. A method of making a viralformulation, comprising the steps of: (a) providing a virus; (b)combining, to form a liquid viral formulation, the virus and a liquidnon-viral composition, wherein the liquid non-viral composition consistsessentially of: (i) sucrose in a concentration of less than 4.5% basedon the weight of the liquid non-viral composition; (ii) Mg²⁺; (iii) anon-ionic surfactant; and (iv) a liquid carrier, wherein the non-viralcomposition is provided in an amount effective to stabilize the virusand wherein monovalent cationic salts are not present in the non-viralcomposition as excipients contributing to the stability of the virus;and (c) lyophilizing the liquid viral formulation, to form a viralformulation.
 30. A method of preserving or stabilizing a virus,comprising: preparing a viral formulation of claim 1; and storing theviral formulation.
 31. The method of claim 30, wherein the virus isstored at a temperature at or below ambient temperature.
 32. The methodof claim 30, wherein the temperature is selected from the groupconsisting of ambient temperature, from 2° C. to 8° C., 4° C., −20° C.,and from −60° C. to −80° C.
 33. A viral formulation of claim 1, whereinthe viral formulation is non-aggregating.
 34. The viral formulation ofclaim 33, wherein the viral formulation is suitable for administrationby parenteral infusion or injection.